Lactobacillus rhamnosus bacterium for treatment of e.g. bacterial vaginosis

ABSTRACT

A novel lactic acid  Lactobacillus rhamnosus  bacterium suitable for treatment of e.g. bacterial vaginosis.

FIELD OF THE INVENTION

The present invention relates to a novel lactic acid Lactobacillusrhamnosus bacterium suitable for treatment of e.g. bacterial vaginosis.

BACKGROUND ART

Bacterial vaginosis (BV) is one of the most common disorders in women ofreproductive age. According to the US Centers for Disease Control andPrevention (CDC), risk factors for BV include douching and having new ormultiple sex partners. BV is caused by an imbalance of naturallyoccurring bacterial microbiota and can be microbiologicallycharacterized by replacement of the lactobacilli-predominant vaginalmicrobiota by potential pathogenic vaginal bacteria. The change from ahealthy, H₂O₂ and lactic acid producing lactobacilli-dominatedmicrobiota to a complex multispecies microbiota can occur relativelyquickly and result in BV.

As discussed in e.g. EP0257007 (filed 1987)—it has been known for longtime that Bacterial vaginosis (BV) is accompanied by a rise in pH (risefrom normal/healthy pH of approximately 4 to a pH above 4.5) and amicrobial dysbiosis in which the usually dominant Lactobacillus vaginalmicrobiota is overwhelmed by an overgrowth of predominantly anaerobicorganisms.

EP2509610B1 (HSO Health Care, Vienna—filed Sep. 13, 2011 andpublished/granted in 2013) provides a summary of herein relevantbackground prior art.

As discussed in EP2509610B1—the healthy human vagina is dominated by avariety of Lactobacillus species, which play an essential role inprotecting women from urogenital infection. Lactobacilli have theability to adhere to vaginal epithelia, to inhibit the adhesion andgrowth of pathogens, deplete nutrients otherwise available to pathogens,and modulate the host immune response and microenvironment. Mostimportantly, Lactobacilli metabolize the glycogen contained in the cellsof the vaginal vault, forming lactic acid as the final product. Thus, ina healthy vagina, pH-values of around 4.0 are reached, a level at whichmany pathogens cannot flourish.

When administrated to a woman in the form of a pharmaceuticalcomposition (e.g. vaginal capsules for vaginal administration)—theprotective effect of Lactobacilli against potential pathogens in thevagina is generated through the metabolic activity of the Lactobacilli.The bacteria consume glycogen and other sources of glucose and producelactic acid. The low pH generated in this manner is harmful to the lessdesirable bacteria and fungi and thus protects the vaginal mucosaagainst infections.

Because vaginal infection is an important mechanism of diseaseresponsible for preterm birth, maintaining the natural, healthy balanceof the Lactobacillus microbiota in the vagina is particularly importantduring pregnancy. A deficiency in Lactobacilli can upset the microbialbalance in the vagina, frequently resulting in the syndrome of bacterialvaginosis, which may be associated with a quantitative and qualitativeshift from normally occurring Lactobacilli to a mixed microbiotadominated by anaerobic bacteria. According to the art, bacterialvaginosis may be characterized by a complete loss of Lactobacilli and aconcomitant increase in Gram-variable and Gram-negative rods, primaryamong them Gardnerella vaginalis as well as Bacteroides, Prevotella, andMobiluncus species. However, loss of vaginal Lactobacilli also leavesnonpregnant women susceptible to infection which may result inendometritis or even pelvic inflammatory disease.

During menopause, involution of the female genital tract occurs,reflecting possibly a built-in biologic life expectancy interrelatedwith the neurohypophyseal endocrine axis. The major universal change isvaginal atrophy. Vaginal dryness, burning, itching and dyspareunia arefrequent complaints along with dysuria, urinary frequency and recurrentinfections. The genitourinary atrophy following menopause is associatedwith a decline in estrogen secretion accompanied by depletion ofLactobacilli and increased colonization by pathogenic microorganismsassociated with bacterial vaginosis and urinary tract infections. Inpost-menopausal women, vaginal estriol therapy reduces Escherichia colicolonization and increases the numbers of Lactobacilli, with the resultthat the incidence of recurrent urinary and genital tract infectionsdrops significantly.

Several species of Lactobacillus have been described to populate thevagina to varying degrees. For some time the microbiota of healthy womenof childbearing age was believed to be dominated by L. acidophilus andL. fermentum, followed by L. brevis, L. jensenii and L. casei. Inanother study on the vaginal Lactobacillus microbiota the authors foundthat the vaginal microbiota of most participants was dominated by asingle Lactobacillus species, with the presence of other species showingwide individual variability. The most frequently occurring species wereL. crispatus, L. gasseri, L. iners, and L. jensenii. In another study,the most commonly isolated Lactobacillus strains were L. jensenii, L.acidophilus, L. casei, and L. gasseri. In recent Austrian studies, thepredominant Lactobacillus species identified by species-specific PCR,namely L. crispatus, L. gasseri, L. jensenii, and L. rhamnosus were usedto generate DNA fingerprints. L. crispatus, L. gasseri, L. jensenii andL. rhamnosus can be regarded as the predominant species in the vagina.

To remedy deficiencies in the Lactobacillus microbiota (and hence, inthe protective capability of the vaginal microbiota), the administrationof vaginal suppositories containing Lactobacilli is the most common wayof Lactobacilli substitution. Some authors believe that the topical useof Lactobacilli is a safe and promising treatment for the prevention ofvaginosis and recurrent urinary tract infections.

While vaginal supplementation is a long-standing, widely acceptedpractice for Lactobacilli substitution, oral administration of aLactobacillus preparation represents a new concept for the restitutionof a normal vaginal microbiota. Relatively recent results indicate thatthe probiotic strains L. rhamnosus GR-1 (ATCC 55826) and L. reuteriRC-14 (ATCC 55845) can be taken orally on a daily basis for two monthswithout any side effects. The consumption then resulted in a significantimprovement of the vaginal microbiota in terms of increased Lactobacillipresence and decreased yeast and coliforms. While one group of authorsdiscussed the beneficial effects in terms of an alteration in mucosalimmunity or of probiotic bacteria ascending to the vagina from therectal area, another group recently demonstrated, by species-specificPCR-amplification, that L. rhamnosus GR-1 and L. reuteri RC-14 can bedelivered to the vaginal environment when administered orally.

In view of above discussed prior art—the invention as described inEP2509610B1 relates to a use of a combination of specific depositedstrains of L. crispatus, L. rhamnosus, L. jensenii and/or L. gasseri fortreatment of bacterial vaginosis (BV). In table 11 on page 15 is shownthat e.g. deposited L. rhamnosus (DSM22560) has a relatively goodcapability to lower pH.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide a novelLactobacillus rhamnosus strain with good properties in particular inrelation to the treatment of vaginal and female genital and urogenitalinfection and/or urinary tract infections caused by Lactobacillusdeficiency (such as e.g. bacterial vaginosis).

As can be seen in working examples below the herein described novel L.rhamnosus strain with the registration number CECT 8800 (herein alsotermed BPL005) has a number of good/improved in particular in relationto the treatment of e.g. bacterial vaginosis.

The results of working Example 1 herein demonstrated following relevantpositive characteristics of the isolated CECT 8800 (=BPL005) L.rhamnosus strain:

1: It was isolated de novo from isolates from vagina of healthywomen—i.e. there is prima facie no reason to believe it is not a novelstrain as such;

2: Random Amplified Polymorphic DNA (RAPD) profile demonstrated that thestrain is different to the others and 16S rRNA sequencing showed thatBPL005 is a unique L. rhamnosus strain;

3: BPL005 had the highest capacity of the vagina isolated strains toreduce pH levels;

4: In assays without pH control BPL005 gave a rapid pH decreasingeffect. The rapid production of lactic acid in the case of BPL005 strainis in agreement with its high capacity to reduce pH in a short time.Regarding the production of the rest of acids, strain BPL005 was thebest producer of the tested strains;

5: With respect to resistance to gynaecological commercialproducts—strain BPL005 was resistant to the highest concentration testedand no inhibitions were observed;

6: Genome sequencing of the whole genome of strain BPL005 showed that ithad a unique contig of 3.023 Mb—i.e. the genome sequence was not a priorart described sequence.

As discussed above—it has been known for long time that Bacterialvaginosis (BV) is accompanied by a rise in pH (rise from normal/healthypH of approximately 4 to a pH above 4.5) and a microbial dysbiosis inwhich the usually dominant Lactobacillus vaginal microbiota isoverwhelmed by an overgrowth of predominantly anaerobic organisms.

Accordingly, the fact that herein described novel CECT 8800 (=BPL005) L.rhamnosus strain has a very good capacity to reduce pH levels isimportant in relation to use of the strain the treatment of e.g.bacterial vaginosis.

In short and without being limited to theory—based of the hereindescribed good properties of the herein described novel CECT 8800(=BPL005) L. rhamnosus strain, there is no reason to believe that itwould not be useful for the treatment of vaginal and female genital andurogenital infection and/or urinary tract infections caused byLactobacillus deficiency (such as e.g. bacterial vaginosis).

The results of working Example 2 herein showed that strain BPL005 hadsimilar pH-reducing levels than the best probiotics tested of hereinrelevant commercial gynaecological probiotic products.

The results of working Example 3 herein showed good efficiency of strainBPL005 in the production process at lab-scale (volume 1 L) and data ofthe made lyophilized powder showed that the BPL005 strain has obtained agood stability in a 15 months period.

The results of working Example 4 herein showed good efficiency of strainBPL005 in the production process at industrial-scale (volume 1500 L) anddata of the made lyophilized powder showed that the BPL005 strain hasobtained a very good stability.

In order to be able to make a herein relevant commercial product it isevident that it is an advantage that the Lactobacillus strain can beproduced properly at industrial scale—accordingly, working Example 4herein demonstrates further advantageous properties of herein describednovel CECT 8800 (=BPL005) L. rhamnosus strain.

Accordingly, a first aspect of the invention relates to a lactic acid L.rhamnosus bacterium with the registration number CECT 8800 (herein alsotermed BPL005) or a mutant thereof, wherein the mutant strain isobtained by using the deposited strain as starting material, and whereinthe mutant has retained or further improved the capacity to reduce pHlevels of CECT 8800.

A second aspect of the invention relates to a dietetic or apharmaceutical composition comprising the bacterium of the first aspectand/or any herein described embodiment thereof.

The term “dietetic” relates according to the art to diet or theregulation of food or feed intake. Accordingly a dietetic composition isa composition that is suitable for being added to a food product for ahuman or a feed product for an animal.

A third aspect of the invention relates to a dietetic or pharmaceuticalcomposition according to the second aspect and/or any herein describedembodiment thereof, for use in the treatment of a disorder in a woman,wherein the disorder is imbalance of naturally occurring vaginalbacterial microbiota, vaginal infection, female genital infection,urogenital infection or urinary tract infection.

A fourth aspect of the invention relates to a dairy product, a drinkproduct, a food product for human nutrition and/or a feed product foranimal nutrition characterized in that the product contains a dieteticcomposition according to the second aspect and/or any herein describedembodiment thereof.

Embodiment of the present invention is described below, by way ofexamples only.

DETAILED DESCRIPTION OF THE INVENTION

Novel Lactobacillus rhamnosus Strain

A sample of the novel Lactobacillus rhamnosus strain BPL005 has beendeposited at CECT (Colección Española de Cultivos Tipo) under theaccession number CECT 8800 with a deposit date of 15^(th) Dec. 2014. Thedeposit has been made under the conditions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

The first aspect of the invention relates to the herein described novelstrain or “a mutant thereof”.

It is clear for the skilled person that by using the deposited strain asstarting material, the skilled reader can by conventional mutagenesis orre-isolation techniques routinely obtain further mutants or derivativesthereof that retain the herein described relevant features andadvantages. Accordingly, the term “a mutant thereof” of the first aspectrelates to mutant strains obtained by using the deposited strain asstarting material.

Alternatively expressed—the invention relates to a method for obtaininga mutant strain of lactic acid L. rhamnosus bacterium with theregistration number CECT 8800 comprising the following steps:

(i): making mutants of the lactic acid L. rhamnosus bacterium with theregistration number CECT 8800;

(ii): analyzing the in step (i) made mutants and identify a mutantstrain that has retained or further improved the capacity of CECT 8800to reduce pH levels; and

(iii) isolating the in step (ii) identified mutant strain to therebyobtain the mutant strain of lactic acid L. rhamnosus bacterium with theregistration number CECT 8800, wherein the mutant strain has retained orfurther improved the capacity of CECT 8800 to reduce pH levels.

Preferably, the first aspect relates to a lactic acid L. rhamnosusbacterium with the registration number CECT 8800 (herein also termedBPL005).

The article (Chenoll E, Codoñer F M, Martinez-Blanch J F, Ramón D,Genovés S, Menabrito M. 2016. Complete genome sequence of Lactobacillusrhamnosus strain BPL5 (CECT 8800), a probiotic for treatment ofbacterial vaginosis. Genome Announc 4(2):e00292-16.doi:10.1128/genomeA.00292-16. Copyright© 2016 Chenoll et al.) waspublished in April, 2016—i.e. after the 29 Jun. 2015 EP15174222.8priority date of the present application.

This article discloses the complete genome sequence of the lactic acidL. rhamnosus bacterium with the registration number CECT 8800 of thepresent invention.

It is evident that it was routine work for the skilled person to obtaina mutant strain of CECT 8800 even without the knowledge of the genomesequence—with the sequence this is even easier.

A Dietetic or a Pharmaceutical Composition—Second Aspect of theInvention

As discussed above—a second aspect of the invention relates to adietetic or a pharmaceutical composition comprising the bacterium of thefirst aspect and/or any herein described embodiment thereof.

The dietetic composition generally comprises dietetic or nutritionalacceptable adjuvants and/or excipients.

The pharmaceutical composition generally comprises pharmaceuticalacceptable adjuvants and/or excipients.

Typically, such composition comprises the bacteria in a concentratedform including frozen, dried or freeze-dried concentrates typicallyhaving a concentration of viable cells, which is in the range of 10⁴ to10¹⁴ cfu (colony forming units) per gram of the composition. It may bepreferred that the range is in the range of 10⁶ to 10¹⁴ cfu (colonyforming units) per gram of the composition or is in the range of 10⁸ to10¹⁴ cfu (colony forming units) per gram of the composition. A relevantrange may also be in the range of 10⁶ to 10¹³ cfu (colony forming units)per gram of the composition.

It may be preferred that the composition comprises freeze-dried(alternatively expressed lyophilized) bacteria.

The composition may as further components contain cryoprotectants (e.g.maltodextrine) and/or conventional additives including nutrients such asyeast extracts, sugars and vitamins.

If the composition comprises freeze-dried bacteria then is preferablycomprises suitable cryoprotectants (e.g. maltodextrine).

As understood by the skilled person in the present context—the dieteticor a pharmaceutical composition as described herein will in certainembodiments comprise a multiplicity of strains either belonging to thesame species or belonging to different species. A typical example ofsuch a useful combination of lactic acid bacteria in a composition asdescribed herein is a mixture of a Leu-conostoc sp. and one or moreLactococcus subsp. such as Lc. lactis subsp. lactis, Lc. lactis subsp.cremoris or Lc.s lactis subsp. lactis biovar. diacetylactis.

It may also be e.g. L. crispatus, L. rhamnosus, Lactobacillus jenseniiand/or L. gasseri.

Application forms of the compositions according to the inventionssuitable for oral intake or topical vaginal administration may bepreferred. As an example, probiotic bacteria for female use can beadministered orally in the form of capsules, or (filled in sachets)suspended in a drink, or in the form of fermented milk (yogurt). Whenadministered orally, they are expected to survive passage through thestomach and duodenum (displaying a certain stability towards acid andbile) and temporarily colonize the gut. From there, small numbers ofbacteria will ascend to the vagina and (again temporarily) colonize thevaginal mucosa. Probiotic bacteria can also be used conventionally asvaginal capsules or suppositories and directly applied to the vagina.The protective effect of Lactobacilli against potential pathogens in thevagina is generated through the metabolic activity of the Lactobacilli.The bacteria consume glycogen and other sources of glucose and producelactic acid. The low pH generated in this manner is harmful to the lessdesirable bacteria and fungi and thus protects the vaginal mucosaagainst infections. Therefore, a composition according to the inventionmay be administered in the form of suppositories, vaginal capsules forvaginal administration or as coated capsules, tablets, sachets, pills,pearls, vials for oral intake as well as yogurt, yogurt drinks,fermented milk, juices, and other fermented drinks and foods.

Use for Treatment of Disorder in a Women—Third Aspect of the Invention

As discussed above—a third aspect of the invention relates to a dieteticor pharmaceutical composition according to the second aspect and/or anyherein described embodiment thereof, for use in the treatment of adisorder in a woman, wherein the disorder is imbalance of naturallyoccurring vaginal bacterial microbiota, vaginal infection, femalegenital infection, urogenital infection or urinary tract infection.

As understood by the skilled person in the present context—in relationto the disorder in a women e.g. a dietetic or pharmaceutical compositionas described herein will many times be used in order to restore and/ormaintain a healthy vaginal bacterial microbiota.

The third aspect may alternatively be formulated as relating to a methodfor treatment of a disorder in a women, wherein the disorder imbalanceof naturally occurring vaginal bacterial microbiota, vaginal infection,female genital infection, urogenital infection or urinary tractinfection, in a human woman comprising administering a relevant amountof a dietetic or pharmaceutical composition according to the secondaspect and/or any herein described embodiment thereof to the humanwoman.

Examples of a herein relevant disorder is vaginosis, vaginitis, chronicbacterial vaginosis, chronic yeast infection, chronic urinary tractinfection in menopause, atrophic vaginitis, vaginosis or bacterialvaginosis.

In a preferred embodiment, the disorder in a woman is bacterialvaginosis (BV).

In addition, the compositions according to the invention areparticularly suitable for use in the treatment or prevention ofasymptomatic and recurrent bacterial vaginosis in pregnancy or pretermdelivery caused by bacterial vaginosis.

Dairy Product, a Drink Product, a Food Product etc. Fourth Aspect of theInvention

As discussed above—a fourth aspect of the invention relates to a dairyproduct, a drink product, a food product for human nutrition and/or afeed product for animal nutrition characterized in that the productcontains a dietetic composition according to the second aspect and/orany herein described embodiment thereof.

Dairy products of the present invention can consist of milk, yogurt,cheese, homogenized products (based on milk, cheese, fruit), fermentedor non-fermented milk (including powdered milk, non-lactose containingmilk, milk shakes) containing probiotics. Therapeutic cheese can beobtained by the addition of suitable probiotic microorganisms in aconcentrated dried form in a certain processing phase of the cheese inorder to guarantee the supply of the dose of the microorganismsnecessary for the organism. The drinks can be instantaneous drinks orwater containing the compositions according to the present invention.

Said integrators, dairy and food products are suitable for use in thetreatment the same disorders in a women as discussed above—i.e. for usein the treatment of a disorder in a women, wherein the disorder isimbalance of naturally occurring vaginal bacterial microbiota, vaginalinfection, female genital infection, urogenital infection or urinarytract infection.

An embodiment of the invention relates to a method of manufacturing afermented food or fermented feed product comprising adding a lactic acidLactobacillus rhamnosus bacterium of first aspect to a food or feedproduct starting material and keeping the thus inoculated startingmaterial under conditions where the lactic acid bacterium ismetabolically active.

Useful food product starting materials include any material which isconventionally subjected to a lactic acid bacterial fermentation stepsuch as milk, vegetable materials, meat products, fruit juices, must,doughs, soya and batters.

The fermented products, which are obtained by the method, include astypical examples dairy products such as fermented milk, yogurt, cheeseincluding fresh cheese products or mozzarella, and buttermilk.

In further embodiments, the substrate material is a starting materialfor an animal feed such as silage, e. g. grass, cereal material, peas,alfalfa or sugar-beet leaf, where bacterial cultures are inoculated inthe feed crop to be ensiled in order to obtain a preservation hereof, orin protein rich animal waste products such as slaughtering offal andfish offal, also with the aims of preserving this offal for animalfeeding purposes.

EXAMPLES Example 1 Isolation and Characterization of L. rhamnosus BPL005of the Invention

Material And Methods

1. Isolation and Selection of the Strains

In order to obtain isolates from vagina of healthy women, samples weretaken from middle area of the vagina of fourteen healthy premenopausalwomen aged between 18 and 45. The exclusion criteria were statedpregnant or lactating women, menstruating at the time of sampling, orwith an antimicrobial therapy against previous vaginitis.

Swabs were spread in MRS agar plates and incubated anaerobically at 37°C. Gram-positive, nonsporulated rods were selected as presumptivelactobacilli. Pure cultures were long-term stored (−20° C.) in glycerolfor further analysis.

2. Molecular Identification

In order to ensure that isolates are unique, agarose Random AmplifiedPolymorphic DNA (RAPD) profiles were generated and duplicate isolateswere rejected.

Molecular identification was carried out by 16S rRNA gene sequencing asChenoll et al. (Novel Probiotic Bifidobacterium bifidum CECT 7366 strainactive against the pathogenic bacterium Helicobacter pylori. 2011.Applied and Environmental Microbiology. 77(4): 1335-1343). Briefly, DNAfrom pure cultures was extracted and purified and 16S rDNA was amplifiedby PCR technology. Amplification products were purified and sequenced.Identification was achieved by comparing sequences obtained with theBLAST database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

3. Selection of the Best pH-Reducing Strain

In order to study the capacity of lactobacilli strains to reduce pHlevels, standardized cultures of the strains were grown in de Man,Rogosa and Sharpe (MRS) medium during 17 h at 37° C. Final pH wasmeasured with the aid of a pH meter. Assays were performed intriplicate. Once experiments performed separately, the combination ofthe most reducing strains to decrease pH levels was tested. To performthis assay, both strains were co-cultivated in MRS medium during 17 h at37° C., and final pH was measured with the aid of a pH meter.

4. Identification of the Organic Acid Profile

To obtain the organic acid profile of selected strains, each strain wascultured anaerobically at 37° C. in 1 L fermentors (Biobundle;Applikon), in the industrial medium optimized in Biopolis forlactobacilli. For each strain, two conditions were assayed: with andwithout pH control. In both cases, levels of pH were monitored duringthe assay. Samples were collected throughout the assay. For organicacids quantification, 0.8 mL of cell-free supernatant was mixed byvortexing with 0.2 mL of a mixture containing 5% meta-phosphoric acid,copper sulfate (1.56 mg/mL) and 50 mM 4-methyl valeric acid as aninternal standard. Samples were then filtered by 0.45 μm pore size anddiluted ½ and 1/10.

The evaluation of organic acids was performed by HPLC chromatography. Analiquot of 10 μL of processed samples was injected onto a HPLC Alliance2695 (Waters) equipped with a Rezex column (ROA Organic Acid (H+) 8%300×7.8 mm, 8 um [Premium]) under conditions defined by manufacturer.Detection was achieved by an index of refraction detector (2414 Waters).The eluent was degassed H₂SO₄ 2.5 mM at an isocratic flow rate of 0.6mL/min. In all cases, quantification curves were constructed adding themixed previously defined.

5. Resistance to Commercial Products.

In order to test the resistance of strain BPL005 to differentgynaecological products, aliquots of direct stocks of each product weredissolved in broth medium to 5120 μg/ml, and 0.22 μm filtered. Stocksolutions were stored at −80° C. From the first dilution, % serialdilutions were performed in a range of 512-0.125 μg/ml. Inoculums wereobtained directly from a 24 h colony, resuspended in saline solution to0.5 MacFarland and diluted 1/20 in saline solution. Assays wereperformed in multiwell plates and incubated anaerobically at 37° C.during 24 h. The Minimum Inhibitory Concentration (MIC) was establishedas the minimum concentration in which the antibiotic exerts a clearinhibition (with no growth). In the case of products with highturbidity, assays included a second step in which 5 μL of 24 h broth ofBPL005 in the IST medium in the presence of serially diluted commercialproducts were dropped in agar plates to confirm the growth of theprobiotic.

6. Genome Sequence

To obtain the whole genome sequence and analysis of the complete genomeof strain BPL005, genome sequencing and bioinformatic analysis are beingperformed.

i) Sequencing:

To obtain the whole genome of the selected strain, a high concentratedpellet was obtained from the master cell bank and high pure DNA wasmanually extracted following Pitcher et al. (Rapid extraction ofbacterial genomic DNA with guanidium thiocyanate. 1989. Letters inApplied Microbiology. 8 (4): 151-156). Sequencing has been performedwith the aid of Pacific Biosciences platform (PacBio).

ii) Bioinformatic Analyses:

Raw sequences obtained from the whole genome sequencing have beenassembled constructing a scaffolding to be used for genome annotation.tRNAs, rRNA s and open Reading Frames (ORFs) will be predicted. ORFsfunctionality will be annotated by comparing each ORF against a databasecontaining all sequences from the bacteria kingdom. Once genefunctionality associated to each ORF, virulence and antibioticresistance genes will be searched and listed.

Results

1. Isolation and Selection of the Strains

In total, fourteen samples were obtained and spread in MRS agar platesin order to obtain isolates. Fourteen strains were obtained out of 100isolates recovered. All of them were obtained from vagina samples. Oncerecovered, strains were long-term stored (−20° C.) in glycerol forfurther analysis. The isolates obtained were classified on the basis ofGram staining and cell morphology. Fourteen Gram-positive,non-sporulated rods were classified as presumptive lactobacilli and thusselected for further identification.

2. Molecular Identification

Once the isolates recovered, agarose RAPD profiles were performed inorder to reject duplicate isolates. On the basis of RAPD profiles, thefourteen isolates were grouped in six different strains. Strains wereidentified by 16S rRNA sequencing (Table 1) and Lactobacillus strainswere selected for further studies (Table 2). In addition, collectionstrains L. crispatus CECT 4840 and L. iners DSM 13335 were included inthe study.

TABLE 1 Groups obtained by RAPD profile and 16S rRNA sequencing. GenusIsolates (strains) Lactobacillus 14 (6) Pathogens 10 (5) “Unculturedbacterium”  6 (6) Others  14 (14)

TABLE 2 Lactobacilli strains obtained. Identification StrainLactobacillus casei BPL013 L. jensenii BPL016 BPL017 BPL035 BPL044 L.rhamnosus BPL005

3.Selection of the Best pH-Reducing Strain

The capacity of strains to reduce pH levels was studied. Table 3summarizes the results obtained. In assays performed with only onestrain, pH levels decreased in all cases. The highest pH reduction wasobtained in the case of strain L. rhamnosus BPL005, being the final pH3.82.

TABLE 3 pH levels obtained in growth cultures of lactobacilli strains(assay with pure cultures). Strain pH Control (medium without culture)6.1 L. crispatus CECT4840 4.55 L. iners DSM13335 4.52 L. casei BPL0134.30 L. jensenii BPL016 4.46 L. jensenii BPL017 4.30 L. jensenii BPL0354.34 L. jensenii BPL044 4.61 L. rhamnosus BPL005 3.82

On the basis of pH reduction results, strain L. rhamnosus BPL005 and L.casei BPL013 were selected for further studies.

4. Identification of the Organic Acid Profile

Organic acids have been described as inhibitory metabolites and also ascompounds with a prebiotic effect. In this sense, strains BPL005 andBPL013 were analysed on the basis of their production of organic acidsin 1 L fermentations. Regarding pH decreasing in assays without pHcontrol—in the case of strain BPL005, pH started decreasing very fast.At 5 hours of fermentation, pH level was 3.7 and at 10 hours offermentation pH reached its minimum (pH 3.0). In the case of BPL013strain, pH decreased gradually, with the lowest pH at 30 hours offermentation.

Tables 4-5 show the production of organic acids. In the case offermentations with pH control, samples were recovered at the end of thefermentation. In all cases, highest levels were obtained for lacticacid, being the highest concentrations 22.53 g/L in the case of BPL013and 19.90 g/L for BPL005 strain, in fermentations with controlled pH.The rapid production of lactic acid in the case of BPL005 strain is inagreement with its high capacity to reduce pH in a short time. Theproduction of lactic acid was followed by a decrease in pH in bothstrains, being more remarkable in the case of strain BPL005.

Regarding the production of the rest of acids, strain BPL005 was thebest producer in all cases. It has been reported that these organicacids could inhibit pathogen bacteria and yeasts (Juarez et al.Urogenital pathogen inhibition and compatibility between vaginalLactobacillus strains to be considered as probiotic candidates. 2011.European Journal of Obstetrics & Gynecology and Reproductive Biology.159: 399-406) In the case of lactic acid, levels obtained were higherthan other previously described in above cited Juárez et al. reference.

TABLE 4 Levels of organic acids (g/L) obtained in BPL005 fermentations.Organic acids concentration (g/L) no pH control pH control 5 h 22 h 45 h22 h Lactic acid 3.172 ± 0.066 12.168 ± 0.223  15.635 ± 0.158  19.731 ±0.232  Acetic acid 0.105 ± 0.050 0.123 ± 0.058 0.143 ± 0.058 0.145 ±0.049 Propionic acid 0.740 ± 0.029 0.155 ± 0.029 0.210 ± 0.026 0.134 ±0.013 Butyric acid 0.074 ± 0.041 0.074 ± 0.041 0.072 ± 0.039 0.082 ±0.041 Succinic acid 0.114 ± 0.025 0.112 ± 0.042 0.157 ± 0.033 0.175 ±0.008

TABLE 5 Levels of organic acids (g/L) obtained in BPL013 fermentations.Organic acids concentration (g/L) no pH control pH control 5 h 30 h 42 h42 h Lactic acid 0.479 ± 0.090 1.387 ± 0.096 10.093 ± 0.088  22.814 ±0.397  Acetic acid 0.060 ± 0.051 0.065 ± 0.051 0.084 ± 0.054 0.109 ±0.064 Propionic acid 0.058 ± 0.041 0.045 ± 0.030 0.058 ± 0.027 0.058 ±0.023 Butyric acid ND ND ND ND Succinic acid 0.092 ± 0.001  0.11 ± 0.0020.104 ± 0.023 0.133 ± 0.032 ND: Not detected.

5. Resistance to Commercial Products Assays.

Results obtained with the gynaecological commercial products aresummarized in Table 6. In all cases, strain BPL005 was resistant to thehighest concentration tested and no inhibitions were observed, even athighest concentrations in products in gel and cream matrix.

TABLE 6 MIC values obtained for progesterone-based products. Product MIC(mg/ml) Ultrogestan >20 mg/ml Progeffik >20 mg/ml Prolutex >20 mg/mlCrinone >22 mg/ml Darstin >2.22 mg/ml*  *Higher concentration was notpossible to test because of its initial concentration.

6. Genome Sequence

The whole genome of strain BPL005 has been sequenced. Whole genomeextraction has been performed; identification of strain corroborated andsequencing has been applied. The assembly rendered a unique contig of3.023 Mb. There were not sequences predicted as plasmids.

Conclusions

The results of this Example demonstrated following herein relevantpositive characteristics of the isolated BPL005 L. rhamnosus strain:

1: It was isolated de novo from isolates from vagina of healthywomen—i.e. there is prima facie no reason to believe it is not a novelstrain as such;

2: Random Amplified Polymorphic DNA (RAPD) profile profile demonstratedthat the strain is different to the others and 16S rRNA sequencingshowed that BPL005 is a unique L. rhamnosus strain;

3: BPL005 had the highest capacity of the vagina isolated strains toreduce pH levels;

4: In assays without pH control BPL005 gave a rapid pH decreasingeffect. The rapid production of lactic acid in the case of BPL005 strainis in agreement with its high capacity to reduce pH in a short time.Regarding the production of the rest of acids, strain BPL005 was thebest producer of the tested strains;

5: With respect to resistance to gynaecological commercialproducts—strain BPL005 was resistant to the highest concentration testedand no inhibitions were observed;

6: Genome sequencing of the whole genome of strain BPL005 showed that ithad a unique contig of 3.023 Mb—i.e. the genome sequence was not a priorart described sequence.

Example 2 Comparison of BPL005 with the Commercial GynaecologicalProbiotics

Material and Methods

Viable Cells in Product.

To study in depth the different commercial probiotic products nowavailable in the market, a collection of 18 probiotics-based productswas analysed. Products tested are summarized in Table 7. For eachproduct, concentration was obtained by counts in MRS plates andfunctionality of strains was tested. To study the functionality,probiotic strains were isolated, identified by 16S rDNA sequencing andits capacity to decrease pH tested.

TABLE 7 Commercial products analysed. Commercial product Kind of productGinegea Candida stick Powder HydralinFlora Capsules mediGYNE CapsulesGynOphilus LP Capsules GynOphilus Capsules FLORGYNAL TAMPON TamponsSymbiovag Capsules SYMBIO Vag Hard vaginal capsule Muvagyn probioticoTampone Muvagyn Probiotico Capsules Isadin α barcilus ISDIN Soft vaginalcapsule NORMOGIN 40 mg Capsules Lactonorm Capsules ACIDIF CV CapsulesVaginol Hard vaginal capsule Gyno-canesflor Capsules Floragyn Hardvaginal capsule Lactoflorene Gyn Hard vaginal capsule

Ability to Reduce pH Levels

Probiotic strains were isolated from commercial products and stored at−80° C. Identification at species level was confirmed by 16S rDNAsequencing. For each strain, pH reduction assays were performed asexplained above. BPL005 strain was included as a control.

Results

Viable Cells in Product

To study in depth the different commercial probiotic products nowavailable in the market, a collection of 18 probiotics-based productswas analysed. Products tested are summarized in Table 8. For eachproduct, concentration was obtained by counts in MRS plates andfunctionality of strains was tested. To study the functionality,probiotic strains were isolated, identified by 16S rDNA sequencing andits capacity to decrease pH tested.

TABLE 8 Comparative analysis of commercial products. Viable Total viableTheoretical viable bacteria Quantity bacteria bacteria Commercialproduct Kind of product (CFU/g) (g) (CFU) (CFU) Ginegea Candida stickPowder 5.70E+09 2 1.14E+10 1.00E+09 HydralinFlora Capsules 2.77E+10 0.205.53E+09 1.00E+08 mediGYNE Capsules 1.30E+09 0.20 2.60E+08 1.00E+08GynOphilus LP¹ Capsules 1.78E+09 0.88 1.56E+09 877 mg¹ GynOphilus¹Capsules 4.00E+09 0.34 1.36E+09 341 mg¹ FLORGYNAL TAMPON Tampons1.18E+09 1.60 1.88E+09 — SYMBIO Vag Hard vaginal capsule 9.00E+08 21.80E+09 1.00E+09 Muvagyn probiotico Tampons 2.20E+07 3.10 6.83E+071.00E+08 Muvagyn Probiotico Capsules 1.80E+09 0.41 7.33E+08 1.00E+08Isadin α barcilus ISDIN Soft vaginal capsule 7.70E+08 2.82 2.17E+091.00E+08 NORMOGIN 40 mg¹ Capsules 3.20E+06 1.00 3.20E+06  40 mg¹Lactonorm Capsules 6.40E+08 0.42 2.70E+08 1.00E+11² ACIDIF CV Capsules1.40E+08 1.05 1.47E+08 5.00E+08 Vaginol³ Hard vaginal capsule — — — —Gyno-canesflor Capsules 2.20E+10 0.34 7.41E+09 1.00E+08 Floragyn⁴ Hardvaginal capsule 5.90E+04 2.00 1.18E+05 — Lactoflorene Gyn Hard vaginalcapsule 2.34E+05 0.62 1.44E+05 1.20E+06 ¹Dosis are given in mg, not inCFU; ²Viables in the time of encapsulation; ³No probiotics in thecomposition; ⁴Lysated probiotics

As shown in Table 8, results were very variable. Some of them showedviable cells lower than specifications.

Ability to Reduce pH Levels

Probiotic strains from commercial products were isolated and theirability to reduce pH levels tested. Table 9 shows the comparisonobtained among them.

TABLE 9 Reduction of pH level obtained for commercial probiotics.Product Probiotics isolated Final pH MediGYNE L. gasserii 4.33 L.rhamnosus 3.55 HydralinFlora L. plantarum 3.55 GynOphilus LP L. caseirhamnosus Döderlein 4.20 FLORGYNAL tampon L. rhamnosus 4.25 L. fermentum4.10 Muvagyn L. gasserii LN40 4.21 L. fermentum LN99 4.23 L. rhamnosusLN113 4.24 Isadin a barcilus ISDIN L. plantarum 3.93 GynOphilus L. caseirhamnosus Döderlein 4.17 Lactonorm L. acidophilus 4.23 BPL005 (control)L. rhamnosus 3.73

Results obtained showed strain BPL005 had similar pH-reducing levelsthan the best probiotics tested.

Conclusions

The results of this Example showed that strain BPL005 had similarpH-reducing levels than the best probiotics tested of herein relevantcommercial gynaecological probiotic products.

Example 3 Evaluation of the Productivity of Lactobacillus Strain BPL005in Laboratory-Scale Production (Volume 1 L)

Material and Methods

For the evaluation of productivity, a master cell bank and a workingcell bank at −80° C. were constructed.

Laboratory-scale production was performed in 1 L fermentor (Biobundle;Applikon), in an industrial medium optimized for lactobacilli. Culturewas obtained in standard conditions (Table 10), and cells were collectedby centrifuging. After collected, pellet was resuspended in amaltodextrine-based carrier solution and frozen at −80° C. andlyophilized.

TABLE 10 Fermentation conditions Parameter Value OD Fermentator t 0 h0.1 Temperature 37° C. Shaking 100 rpm Atmosphere Anaerobiosis pH 6.3Culture medium BRFS

In order to evaluate the resistance of the strain to freezing andlyophilisation processes, cell viability was checked on MRS agar platesbefore being collected and after lyophilisation.

Study of Stability of Lyophilized Product

The lyophilized powder was vacuum-packaged and stored at 5° C. (±3° C.).Stability of the product was checked by counts in MRS during 15 months.

Results

In order to obtain the evaluation of strain BPL005 efficiency, alab-scale production was performed. Results obtained are summarized inTable 11. Results obtained showed a good efficiency of strain BPL005 inthe production process at lab-scale.

TABLE 11 Results obtained after 1 L fermentation. Parameter Value Timeof harvest 20 h Final OD 8.1 Final CFU/ml in fermentor 9 × 10⁹ CFU/mlFinal CFU/g in lyophilized 2.7 × 10¹² CFU/g

The lyophilized powder was vacuum-packaged and stored at 5° C. Stabilityof the product was checked by counts in MRS. The BPL005 strain hasobtained a good stability in a 15 months period.

Conclusions

The results of this Example showed good efficiency of strain BPL005 inthe production process at lab-scale (volume 1 L) and data of the madelyophilized powder showed that the BPL005 strain has obtained a goodstability in a 15 months period.

Example 4 Industrial Production of Lactobacilli BPL005 Strain (Volume1500 L)

Material and Methods

Industrial Scale-Up Production

Industrial production was carried out in a 1500 L fermentor. Growth wasperformed in an industrial medium optimized for lactobacilli. Culturewas obtained in standard conditions (anaerobically, controlling pH andtemperature), and cells were collected by centrifuging in an industrialcentrifuge.

Optimization of Lyophilisation

Once obtained, pellet was resuspended in carrier (maltodextrine-based)and lyophilised in an industrial lyophilizer. To obtain the capacity ofthe strain to resist lyophilisation process, viability was checked onMRS agar plates just after fermentation process and after industrialfreeze-drying process. The purity of culture and lyophilised product waschecked by electrophoretic RAPD (Random Amplification of PolymorphicDNA) profiles of randomly selected colonies.

Shelf-Life Evaluation of Bulk Powder.

Shelf-life of bulk powder is in study. Aliquots of bulk-powder arestored under 5° C. and 25° C. Evaluation will be performed periodicallyby counts on MRS agar.

Results

Industrial Scale-Up Production

Industrial production was carried out in a 1500 L fermentor followingstandard conditions. Lactobacillus rhamnosus BPL005 culture rendered6.55×10⁹ CFU/mL. This result is in agreement with results obtained in 1L scale, and acceptable in terms of efficiency of the process.

Optimization of Lyophilisation

Counts obtained are summarized in Table 12. Results obtained showed agood efficiency of strain BPL005 to the production process at 1500 Lscale.

TABLE 12 Results obtained after 1500 L fermentation. Time of harvest 17h Final CFU/ml in fermentor 6.55 × 10⁹ CFU/ml Final CFU/g in lyophilized5.53 × 10¹¹ CFU/g

Shelf-Life Evaluation of Bulk Powder.

The stability obtained in the 1500 L production at 5° C. and 25° C. ofthe vacuum-packed lyophilized strain BPL005 showed that the product wasvery stable at a temperature of 5° C. Results obtained at 25° C. show anacceptable low decrease in numbers.

Conclusions

The results of this Example showed good efficiency of strain BPL005 inthe production process at industrial-scale (volume 1500 L) and data ofthe made lyophilized powder showed that the BPL005 strain has obtained avery good stability.

REFERENCES

1: EP0257007 (filed 1987)

2: EP2509610B1 (HSO Health Care, Vienna—filed Sep. 13, 2011 andpublished/granted in 2013)

3: Chenoll E, Codoñer F M, Martinez-Blanch J F, Rannón D, Genovés S,Menabrito M. 2016. Complete genome sequence of Lactobacillus rhamnosusstrain BPL5 (CECT 8800), a probiotic for treatment of bacterialvaginosis. Genome Announc 4(2):e00292-16. doi:10.1128/genomeA.00292-16.Copy-right© 2016 Chenoll et al.—published in April, 2016

1. A lactic acid Lactobacillus rhamnosus bacterium with the registrationnumber CECT
 8800. 2. A composition comprising the bacterium of claim 1.3. The composition of claim 2, wherein the composition is having aconcentration of viable cells, which is in the range of 10⁴ to 10¹⁴ cfu(colony forming units) per gram of the composition, preferably whereinthe concentration of viable cells is in the range of 10⁶ to 10¹³ cfu(colony forming units) per gram of the composition.
 4. The compositionof claim 2, wherein the composition comprises the bacteria in a frozenform, dried form or freeze-dried form.
 5. The composition of claim 4,wherein the composition comprises the bacteria in freeze-dried form andthe composition also comprises suitable cryoprotectants (e.g.maltodextrine).
 6. The composition of claim 2, wherein the compositionis suitable for oral intake or vaginal administration.
 7. Thecomposition of claim 6, wherein the composition is in the form ofsuppositories, vaginal capsules for vaginal administration or as coatedcapsules, tablets, sachets, pills, pearls, vials, for oral intake,yogurt, yogurt drinks, fermented milk, juices, and other fermenteddrinks and foods.
 8. The dietetic or a pharmaceutical composition ofclaim 2, wherein the composition is a pharmaceutical composition thatalso comprises pharmaceutical acceptable adjuvants and/or excipients. 9.A method of treating a disorder in a woman, wherein the disorder isimbalance of naturally occurring vaginal bacterial microbiota, vaginalinfection, female genital infection, urogenital infection or urinarytract infection comprising administering to a woman in need thereof anamount of the composition of claim 1 effective to treat said disorder.10. The composition of claim 9, wherein the disorder is vaginosis,vaginitis, chronic bacterial vaginosis, chronic yeast infection, chronicurinary tract infection in menopause, atrophic vaginitis, vaginosis orbacterial vaginosis.
 11. The composition of claim 10, wherein theinfection is bacterial vaginosis.
 12. A dairy product, a drink product,a food product for human nutrition and/or a feed product for animalnutrition characterized in that the product contains the compositionaccording to claim
 2. 13. A method of manufacturing a fermented food orfermented feed product comprising adding a lactic acid Lactobacillusrhamnosus bacterium of claim 1 to a food or feed product startingmaterial and keeping the thus inoculated starting material underconditions where the lactic acid bacterium is metabolically active. 14.A method for obtaining a mutant strain of lactic acid Lactobacillusrhamnosus bacterium with the registration number CECT 8800 comprisingthe following steps: (i): making mutants of the lactic acidLactobacillus rhamnosus bacterium with the registration number CECT8800; (ii): analyzing the in step (i) made mutants and identify a mutantstrain that has retained or further improved the capacity of CECT 8800to reduce pH levels; and (iii) isolating the in step (ii) identifiedmutant strain to thereby obtain the mutant strain of lactic acidLactobacillus rhamnosus bacterium with the registration number CECT8800, wherein the mutant strain has retained or further improved thecapacity of CECT 8800 to reduce pH levels.